Leaf Plasmodesmata Respond Differently to TMV, ToBRFV and TYLCV Infection

Abstract

Macromolecule and cytosolic signal distribution throughout the plant employs a unique cellular and intracellular mechanism called plasmodesmata (PD). Plant viruses spread throughout plants via PD using their movement proteins (MPs). Viral MPs induce changes in plasmodesmata's structure and alter their ability to move macromolecule and cytosolic signals. The developmental distribution of a family member of proteins termed plasmodesmata located proteins number 5 (PDLP5) conjugated to GFP (PDLP5-GFP) is described here. The GFP enables the visual localization of PDLP5 in the cell via confocal microscopy. We observed that PDLP5-GFP protein is present in seed protein bodies and immediately after seed imbibition in the plasma membrane. The effect of three different plant viruses, the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV, tobamoviruses), and tomato yellow leaf curl virus (TYLCV, begomoviruses), on PDLP5-GFP accumulation at the plasmodesmata was tested. In tobacco leaf, TMV and ToBRFV increased PDLP5-GFP amount at the plasmodesmata of cell types compared to control. However, there was no statistically significant difference in tomato leaf. On the other hand, TYLCV decreased PDLP5-GFP quantity in plasmodesmata in all tomato leaf cells compared to control, without any significant effect on plasmodesmata in tobacco leaf cells.

Keywords: TMV; TYLCV; ToBRFV; plant viruses; plasmodesmata; tobacco mosaic virus; tomato brown rugose fruit virus; tomato yellow leaf curl virus.

Figure 1

Distribution of PDLP5-GFP tobacco leaf cells. (a) Close-up of the tobacco cell wall between adjacent epidermis cells, (b) close-up of tobacco mesophyll cell pit field, the red globes are chloroplasts. (c) Close-up of stomata in tobacco leaf, (d) PD in immature tomato seeds, (e) tomato mesophyll cells, (f) stomata, and the base of trichomes in tomato leaf. A white arrow marks the cell wall, red arrow pit fields, yellow arrow points to a single PD, and a blue arrow points to pit fields at the base of trichomes. The bar shows the distance in µm.

Figure 2

Distribution of PDLP5-GFP in dry seeds of tomato and tobacco. (a) Whole dry seed of tobacco, (b) close-up of dry tobacco root, (c) close-up of dry tobacco cotyledon, (d) close-up of dry storage bodies in tobacco root, (e) close-up of dry tomato root, (f) close-up of dry tomato cotyledon. The bar shows the distance in µm.

Figure 3

Distribution of PDLP5-GFP in imbibed seeds of tobacco. (a) Cotyledon-showing disappearance of PDLP5-GFP from outer cell layer after two days of imbibition. Bar = 50 µm (b) close-up of root cells in four days imbibed tobacco seed showing the presence of PDLP5-GFP in the cell wall. Bar = 10 µm (c) close-up of root tip cells in two days imbibed tobacco seed. Bar = 10 µm. The bar shows the distance in µm. GB = granular body; PD = plasmodesmata.

Figure 4

PDLP5-GFP is present in all surface cells of tobacco and tomato 7-day seedlings. The bar shows the distance in µm. (a) Meristematic area of 7 days old tobacco seedling where the new leaf emerge between the cotyledons; (b) Meristematic area of 10 days old tomato seedling where the new leaves emerge between the cotyledons.

Figure 5

AtPDLP5-GFP is localized to the cell wall of all tobacco and tomato cells. (a) Root radical epidermis; (b) pit fields in leaf vain cells; (c) trichomes; (d) leaf epidermis cells; (e) very young tomato fruit pericarp cells; and (f) tomato pistil cells. The bar shows the distance in µm.

Figure 6

Fluorescence intensity of PDLP5-GFP protein in various cells in leaves of tomato and tobacco plants. Tomato and tobacco plants were transformed with 35S::PDLP5-GFP and were viewed in confocal microscopy. The intensity of GFP fluorescence in the plasmodesmata decorated with GFP per µm of the cell wall was measured per cell type. In each graph, means (± SE) are shown. Different letters above the data columns mean that differences between treatments are statistically significant (p{f} <  0.001). Each genotype was tested separately.

Figure 7

Fluorescence intensity of PDLP5-GFP protein in various cells after TYLCV and TMV infection in leaves of tobacco and tomato plants. Tobacco (a) and tomato (b) plants were stably transformed with 35S::PDLP5-GFP and were viewed in confocal microscopy 30 days after inoculation with TYLCV (TY) or TMV (TMV) or mock-inoculated (control). (a,b) Fluorescence area in the GFP per µm of the cell wall was measured per cell type. In each graph, means (± SE) are shown. Different letters above the data columns mean that differences between treatments are statistically significant (p{f} <  0.001), and each cell type was tested separately.

Figure 8

Fluorescence intensity of PDLP5-GFP protein in epidermis cells after ToBRFV and TMV infection in leaves of tomato plants (money maker). Tomato plants were stably transformed with 35S::PDLP5-GFP and were viewed in confocal microscopy 30 days after inoculation with ToBRFV (TBR) or TMV (TMV) or mock-inoculated (control). (a–c) pictures show GFP and chlorophyll fluorescence in various cell types; (d) fluorescence area of GFP or chlorophyll per µm of the cell wall was measured per treatment. Means (± SE) are shown. Different letters above the data columns mean that differences between treatments are statistically significant (p{f} < 0.001) in the parameter analyzed.